The goal of this project is to establish a generic approach, biotin and ubiquitin (Ub) labeling of ligase substrates (BULLS), for defining specific substrates of individual ubiquitin ligases in vivo. Specific substrates will be modified by biotin and ubiquitin simultaneously during the substrate-E3 interaction, enabling subsequent affinity purification and proteomics identification. Ubiquitin, an essential small protein in eukaryotic cels, regulates nearly all cellular events, and its role in neurodevelopment and neurological pathogenesis is widely appreciated. This reversible posttranslational modification is initiated by a cascade of enzymatic reactions through E1/E2/E3 enzymes, and is removed by deubiquitinating enzymes (DUBs). The human proteome contains two Ub E1 enzymes, ~40 different E2s, at least 600 E3 ligases and ~95 DUBs. The specificity in Ub signaling is primarily attributed to selective modification of protein substrates by diverse E3 ligases. Genetic and biochemical studies reveal that a growing list of Ub ligases plays critical roles in neurodevelopment, and that Ub dysregulation contributes to neurological disorders. The investigation of underlying molecular mechanisms, however, is hampered by the lack of sensitive tools to identify corresponding substrates of these Ub ligases. We have developed a series of mass spectrometry (MS)-based technologies to analyze the ubiquitinated proteome (ubiquitinome), with a focus on the role of Ub signaling in neurodevelopment. Here we propose to invent a dual labeling approach of BULLS for identifying specific substrates modified by individual E3 enzymes. We will explore this idea using the E3 ligase mind bomb 1 (Mib1) that functions in neuronal morphogenesis. Two specific aims are: (i) to set up an efficient approach for in vivo biotin labeling and in vitro purification of specific substrates modified by a ligase, nd (ii) to systematically identify Mib1 substrates in neurons by quantitative proteomics. Once developed, we anticipate that this strategy will become a simple and sensitive approach for determining the E3-substrate connection. PUBLIC HEALTH RELEVANCE: Ubiquitin regulates nearly all cellular events including neurodevelopment, synaptic plasticity, learning and memory. We propose to develop a novel, in vivo labeling method to identify specific protein substrates modified by ubiquitin ligases. The method will provide a generic approach for establishing the connection map between ubiquitin ligases and substrates, promoting the investigation of ubiquitin signaling in neuroscience.